Lawsonia intracellularis of European origin and vaccines, diagnostic agents and methods of use thereof

ABSTRACT

The present invention relates to  Lawsonia intracellularis  vaccines and methods for protecting against and diagnosing  L. intracellularis  infection. The products and processes of the invention are attainable, in part, as the result of an improved method for cultivating large scale supplies of  L. intracellularis , including both a novel isolate of  L. intracellularis  of European origin and a method of preparing a lyophilized product containing the attenuated European isolate as vaccine product.

RELATED APPLICATION

This application claims benefit to U.S. Provisional Application Ser. No.60/490,001, filed Jul. 25, 2003, the disclosure of which is incorporatedby reference in its entirety.

BACKGROUND

The present invention relates to Lawsonia intracellularis vaccines andmethods for protecting against and diagnosing L. intracellularisinfection. The products and processes of the invention are attainable,in part, as the result of an improved method for cultivating large scalesupplies of L. intracellularis, including both a novel isolate of L.intracellularis of European origin and a method of preparing alyophilized product containing the attenuated European isolate asvaccine product.

L. intracellularis, the causative agent of porcine proliferativeenteropathy (“PPE”), affects virtually all animals, including: rabbits,ferrets, hamsters, fox, horses, and other animals as diverse asostriches and emus. L. intracellularis is a particularly great cause oflosses in swine herds in Europe as well as in the United States.

A consistent feature of PPE is the occurrence of intracytoplasmic,non-membrane bound curved bacilli within enterocytes in affectedportions of intestine. The bacteria associated with PPE have beenreferred to as “Campylobacter-like organisms.” S. McOrist et al., Vet.Pathol., Vol. 26, 260-264 (1989). Subsequently, the causative bacteriahave been identified as a novel taxonomic genus and species,vernacularly referred to as Ileal symbiont (IS) intracellularis. C.Gebhart et al., Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3,533-538 (1993). More recently, these novel bacteria have been given thetaxonomic name Lawsonia (L.) intracellularis. S. McOrist et al., Int'l.J. of Systemic Bacteriology, Vol. 45, No. 4, 820-825 (1995). These threenames have been used interchangeably to refer to the same organism asfurther identified and described herein.

L. intracellularis is an obligate, intracellular bacterium which cannotbe cultured by normal bacteriological methods on conventional cell-freemedia and has been thought to require attached epithelial cells forgrowth. S. McOrist et al., Infection and Immunity, Vol. 61, No. 19,4286-4292 (1993) and G. Lawson et al., J. of Clinical Microbiology, Vol.31, No. 5, 1136-1142 (1993) discuss cultivation of L. intracellularisusing IEC-18 rat intestinal epithelial cell monolayers in conventionaltissue culture flasks. In addition, H. Stills, Infection and Immunity,Vol. 59, No. 9, 3227-3236 (1991) discusses using Intestine 407 humanembryonic intestinal cell monolayers and GPC-16 guinea pig colonicadenocarcinoma cell monolayers in conventional tissue culture flasks.

Recently, an L. intracellularis vaccine has been approved for use in theUnited States, which vaccine is based on L. intracellularis isolatesdescribed and claimed in U.S. Pat. Nos. 5,714,375 and 5,885,823, both ofwhich patents are herein incorporated by reference in their entireties.The above-described vaccine is sold by Boehringer Ingelheim Vetmedica,Inc., 2621 North Belt Highway, St. Joseph, Mo. 64506-2002, under thetrademark ENTERISOL® Ileitis.

SUMMARY OF THE INVENTION

One object of the invention is to provide an improved L. intracellularisvaccine using an isolate of European origin.

Another object of the invention is to provide an improved method forcultivation of L. intracellularis on a large scale and improvedtechniques for production of L. intracellularis vaccines.

To achieve these and other objects, and in accordance with the purposeof the invention as embodied and broadly described herein, the presentinvention provides a newly isolated L. intracellularis from Europe, amethod of attenuating such an isolate, and the attenuated isolatethereof. Also provided herein is a vaccine comprising the attenuatedisolate. Also provided herein is a method for producing a vaccinecomprising the attenuated isolate in lyophilized form for reconstitutionat the time of administration and the lyophilized product thereof.

In one embodiment, the newly isolated L. intracellularis from Europe,isolate DK 15540, is deposit isolate ATCC accession No. PTA-4927. Inanother embodiment, the attenuated isolate derived from isolate DK15540, is designated isolate B3903, ATCC accession No. PTA-4926.

DETAILED DESCRIPTION

As used herein, the term “L. intracellularis” means the intracellular,curved gram-negative bacteria described in detail by C. Gebhart et al.,Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3, 533-538 (1993) andS. McOrist et al., Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4,820-825 (1995), each of which is incorporated herein by reference intheir entireties, and includes but is not limited to the isolatedesignated DK 15540 which was deposited under the Budapest Treaty withthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209 on Jan. 9, 2003 and assigned ATCC accessionnumber PTA-4927; the causative bacteria which can be obtained from PPEinfected swine or other animals throughout the world given the knowledgein the art and the teachings herein; and variants or mutants of any ofthe above bacteria, whether spontaneously or artificially obtained.

As used herein, the term “attenuated isolate” means any L.intracellularis isolate that is prepared according to the cultivationand passaging techniques taught herein to achieve avirulence whilemaintaining immunogenic properties when administered to a host animalincluding but not limited to the attenuated isolate designated B-3903which was deposited under the Budapest Treaty with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209on Jan. 9, 2003 and assigned accession number PTA-4926.

The attenuated isolate of the invention can be used as an immunogen inantimicrobial vaccines for animals, including birds, fish, and mammalssuch as cattle, swine, horses, and primates. Such vaccines can beprepared by techniques known to those skilled in the art and given theteachings contained herein. Such a vaccine would comprise animmunologically effective amount of the attenuated isolate in apharmaceutically acceptable carrier. The vaccine could be administeredin one or more doses. An immunologically effective amount is determinedby means known in the art without undue experimentation, given theteachings contained herein. The amount of avirulent bacteria should besufficient to stimulate an immune response in disease-susceptibleanimals while still being avirulent. This will depend upon theparticular animal, bacteria, and disease involved. The recommended doseto be administered to the susceptible animal is preferably about 3.0TCID₅₀ (tissue culture infective dose 50% end point)/dose to about 6.0TCID_(5.0)/dose and more preferably about 4.0 TCID₅₀/dose to about 5.0TCID₅₀/dose. In a preferred embodiment, the titer of the vaccine isabout 4.9 TCID₅₀/dose as determined by Tissue Culture Infective Dose 50%endpoint dilution assay (TCID₅₀). The carriers are known to thoseskilled in the art and include stabilizers and diluents. Such a vaccinemay also contain an appropriate adjuvant. The vaccines of the inventionmay be used in combination with other vaccines, for example, as adiluent of another vaccine. The vaccine preparations may also bedesiccated, for example, by freeze drying for storage purposes or forsubsequent formulation into liquid vaccines.

Accordingly, the invention also comprises a method for inducing animmune response to virulent, wild-type L. intracellularis bacteria in ananimal host for the purpose of protecting the host from such bacteria.The method comprises administering an immunologically effective amountof the attenuated bacteria or killed bacteria of the invention to thehost and, preferably, administering the vaccine of the invention to thehost.

As used herein, the term “large-scale cultivation” means a level ofcultivation of L. intracellularis greater than approximately 2.0 to 3.0liters and includes production on a scale of 100 liters or more.“Cultivation” as used herein, means the process of promoting the growth,reproduction and/or proliferation of L. intracellularis.

L. intracellularis can be cultivated by methods known in the art,preferably, according to U.S. Pat. Nos. 5,714,375 and 5,885,823. Forexample, culture cells may first be inoculated with an inoculumcomprising L. intracellularis bacteria so as to infect the cells withthe bacteria. Numerous cell lines can be used in practicing theinvention, including, but not limited to, IEC-18 (ATCC 1589)-ratintestinal epithelial cells, HEp-2 (ATCC 23)-human epidermoid carcinomacells, McCoys (ATCC 1696)-mouse (non-specified) cells, BGMK(Biowhittaker #71-176)-buffalo green monkey kidney cells, and swineintestinal epithelium cells. The preferred culture cells are HEp-2,McCoys or IEC-18 cells.

If culture cells are used, prior to being inoculated, the cells may bein the form of a monolayer. To form a monolayer, the cells may be seededinto conventional flasks. Each flask is generally seeded with betweenabout 1×10⁵ cells to about 10×10⁵ cells per 25, 75, 150, 850 cm² flaskor roller bottle mixed with growth media. The growth media may be anymedia for cell cultivation which includes a nitrogen source, necessarygrowth factors for the chosen culture cells, and a carbon source, suchas glucose or lactose. The preferred media is DMEM fortified with Ham'sF 12 with 1-5% fetal bovine serum, although other commercially availablemedia may be used with good results.

Successful cultivation of L. intracellularis is enhanced by maintainingthe culture cells in a constant state of growth. Therefore, the culturecell monolayer should be at about 20 percent to about 50 percentconfluency at the time of inoculation. Preferably, the cells should beat about 30 percent to about 40 percent confluency at the time ofinoculation, most preferably at about 30 percent confluency.

Alternatively, the cells, prior to being inoculated, may be grown insuspension, as described infra. Preferably, the cells are first grown to100% confluency in the form of a monolayer in an adherent type system,e.g., a roller bottle system, and then transferred to 3-3000 liters andgrown in suspension. Alternatively, the cells can be grown in suspensionto the desired cell density, e.g. 2×10⁵ cells/ml, within the 3-3000liter vessel (bioreactor, Fermentor, spinner flask, etc.) usingparameters suitable for growth within this system prior to inoculation.

The inoculum may be a pure culture of L. intracellularis obtained frominfected swine or other animals. Preferably the inoculum may be a pureculture of L. intracellularis obtained from ATCC accession No. PTA-4927.

The inoculum can be an intestinal homogenate prepared by scraping themucosa off of the ileum of a swine or other animal infected with PPE.When preparing an intestinal homogenate, ileal sections selected forculture should show severe lesions with gross thickening of the gut. Dueto the fragile nature of the bacteria, samples should preferably bestored at −70° C. as quickly as possible after necropsy. An antibioticto which L. intracellularis is resistant such as Vancomycin,Amphotericin B or members of the aminoglycoside group of antibiotics,including Gentamicin and Neomycin, to name a few, is preferably added tothe inoculum to suppress contaminating bacteria while permitting L.intracellularis growth. Whether the inoculum is a pure culture or anintestinal homogenate, inoculation of the culture cells can be performedby various techniques known in the art, given the teachings herein.

The bacteria and/or inoculated culture cells are then incubated under areduced dissolved O₂ concentration. At dissolved oxygen concentrationsgreater than 10% L. intracellularis growth is less than optimal withcessation of growth eventually occurring at oxygen concentrationsoutside this range. Preferably, the bacteria and/or inoculated culturecells are incubated in a dissolved oxygen concentration in the range ofabout 0% to about 10%. More preferably, the bacteria and/or cells areincubated in an oxygen concentration in the range of about 0% to about8%, with an oxygen concentration of about 0% to about 3.0% being mostpreferred.

The proper concentration of carbon dioxide is also important to theproper growth of L. intracellularis. At carbon dioxide concentrationsgreater than 0% and less than 4%, non-optimum growth occurs withcessation of growth eventually occurring at carbon dioxideconcentrations outside this range. Preferably, the carbon dioxideconcentration is in the range from about 6% to about 10%, with a carbondioxide concentration of about 8.8% being most preferred.

In addition, the cells are preferably incubated at a hydrogenconcentration in the range from about 4% to about 10%. Most preferably,the cells are incubated in about 0 to about 8.0% O₂, about 8.8% CO₂, andabout 4% H₂. Nitrogen is used as a “balance” in the gas mixturecontaining nitrogen (96%) and hydrogen (4%) or nitrogen (80%), carbondioxide (10%) and hydrogen (10%) for growth of this organism. Cells arepreferably incubated at a nitrogen concentration in the range from about80% to 96%. Therefore, cells are most preferably incubated in about 0 toabout 8.0% O₂, about 8.8% CO₂, about 4% H₂ and about 96% N₂.

Inoculated cells may be incubated in a dual gas incubator or other gaschambers which contain the proper hydrogen, oxygen and carbon dioxideconcentrations and which allow the cells to be suspended duringincubation. The chamber should comprise a means for maintaining theinoculated cells in suspension, and a gas monitor and supply source tosupply and maintain the proper gas concentrations. The incubationtemperature should be in the range of from 30° C. to about 45° C. and ismore preferably in the range of from about 36° C. to about 38° C. Mostpreferably, the temperature is about 37° C. The necessary equipment forcultivation and attenuation is readily available to those of ordinaryskill in the art given the teachings herein. One example of equipmentsuitable for carrying out the present invention is a dual gas incubator,e.g., model 480 (Lab-Line, Melrose Park, Ill.) in conjunction withspinner flasks to maintain the cells in suspension. The presentlypreferred equipment comprises a fermentor, bioreactor, stir plate orrotary shaker containing at least about 2 liters media and capable ofmaintaining the culture cells in suspension via sparging gas of theappropriate concentration, or other means of mechanical agitation, andcontinuously monitoring dissolved O₂ levels in the media. New Brunswick,Braun and other companies make suitable fermentors and bioreactors forthis purpose.

By maintaining the inoculated cells in a suspended state duringincubation, maximum growth of the cells, and hence L. intracellularis,is achieved by increasing each individual cell's exposure to growthmedia and the proper mixture of hydrogen, oxygen and carbon dioxide. Theculture cells can be agitated and maintained in suspension by a varietyof methods known in the art including, for example, culture flasks,roller bottles, membrane cultures, biobags, WAVE® bioreactor systems,fermentors and spinner flasks. The cells may be kept in suspensionduring incubation by incubating the cells in a spinner flask inside adual gas incubator or similar apparatus. The term “spinner flask”, asused herein, means a flask or other container which employs a paddle,propeller or other means to agitate the culture and keep the cellscontained therein in suspension.

In a preferred embodiment, the inoculated cells are incubated until thecells reach confluency and then the cells are placed in a spinner flaskcontaining growth media and incubated in a dual gas incubator whilespinning the flask. Preferably, the inoculated cells are scraped ortrypsinized and passaged into the spinner flask. This can be achieved bya variety of methods known in the art such as using a cell scraper todetach the cells. Once the cells are introduced into the spinner flask,the paddle of the spinner flask is typically rotated in the range offrom about 5 to about 500 rpm on a magnetic stir plate in order tomaintain the infected cells in suspension.

A portion of the cultivated L. intracellularis is then passaged to freshculture to increase the production of L. intracellularis bacteria. Theterm “passaging” or variations thereof herein means the process oftransferring a portion of the cultivated L. intracellularis to freshculture cells in order to infect the fresh cells with the bacterium. Theterm “fresh”, as used herein, means cells which have not yet beeninfected by L. intracellularis. Preferably such cells are on the averageno more than approximately one day old.

The passage of L. intracellularis in suspension cultures may beaccomplished by removing a portion of the original culture and adding itto a new flask containing fresh culture cells. If the original culturehas a high number of bacteria/ml, for example, greater than about 10⁴bacteria/ml, it is preferable to add between about 1 to 10% (volume tovolume) of culture from the infected flask to a new flask containingfresh cells. This is preferably done when 50-100% of the cells areinfected. If fewer than 50% of the cells are infected, passaging ispreferably accomplished by splitting the culture 1:2 into a new flaskand scaling-up the volume by adding fresh tissue culture cells andmedia. In either case, cell lysis and other steps are not required, indirect contrast to the passage of monolayer cultures, as in the priorart.

After sufficient growth of the culture cells and subsequent infection byL. intracellularis, as determined by indirect fluorescent antibody (IFA)staining, TCID₅₀ or another comparable method, at least a portion of thecultivated L. intracellularis bacteria is then harvested. Harvesting istypically performed at cell infectivity of about 60% or higher; however,one skilled in the art knows that harvesting could be performed at acell infectivity of less than 60%. The harvesting step may be performedby separating the bacteria from the suspension by various techniquesknown to those of ordinary skill in the art, given the teachings herein.Preferably, the L. intracellularis bacteria is harvested by centrifugingthe contents of all or a portion of the suspension to pellet the culturecells, resuspending the resulting cell pellets, and lysing the infectedcells. Typically, at least a portion of the contents is centrifuged atabout 3000×g for about 20 minutes in order to pellet the cells andbacteria. The pellet may then be resuspended in, for example, asucrose-phosphate-glutamate (SPG) solution and passed approximately 20times through a 25 gauge needle in order to lyse the cells. If furtherpurification is desired, the samples can be centrifuged at about 145×gfor about five minutes to remove cellular nuclei and debris. Thesupernatant may then be centrifuged at about 3000×g for about twentyminutes and the resulting pellet resuspended in an appropriate diluent,such as SPG with fetal bovine serum (to prepare harvested bacteriasuitable for lyophilization, freezing, or use as an inoculant) or growthmedia (to prepare harvested bacteria more suitable for passaging tofresh cells).

As previously mentioned, effective growth of L. intracellularis forlarge-scale production is enhanced by keeping the tissue cells activelygrowing. With monolayers, when cultures become confluent, the rate ofcell division decreases substantially. Attempts to grow L.intracellularis on monolayer tissue cultures have had limited successand scale-up has not been possible. However, using suspension culturesgreatly facilitates keeping the cells actively growing and permitscontinuous culture expansion and scale-up. Using a fermentor and betweenabout 0 to 3% dissolved O₂ as explained above, enables growth of up toand greater than 10⁸ bacteria/ml.

When using IEC-18 cells, it is preferable to add gelatin, agarose,collagen, acrylamide or silica beads, such as Cultisphere-G porousmicrocarriers (HyClone Laboratories, Logan Utah), along with the growthmedia. However, HEp-2 cells and others do not require microcarriersaccording to the methods used herein.

For culture maintenance purposes, with HEp-2 cultures, preferably 25% to50% of the culture is removed and replaced with fresh media at weeklyintervals. For cell cultures with microcarriers or beads, preferably 25%to 50% of the culture is removed and replaced with fresh media 1-2 timesweekly. For scale-up purposes, an additional 25% to 50% of media, ormedia with microcarriers, may be added to the culture.

Depending upon the rate at which the culture cells become infected,passage to fresh cells generally occurs between about every 2 to about 7days. Assuming that the culture cells become at least 70% infectedwithin 2 to 7 days, preferably passage occurs between about every 5 to 7days.

The present invention also provides vaccines and methods for producingvaccines against a novel isolate of L. intracellularis of Europeanorigin. Preferably, after maintaining the infected cells in suspensionfor an extended time (for example, 6-8 months), at least a portion ofthe cultivated L. intracellularis bacteria are harvested and monitoredfor potential attenuation. Such monitoring is preferably accomplished byhost animal or animal model challenges to select for an attenuatedisolate. Such attenuated isolates are used in vaccines according to themethods taught herein.

The present invention allows rapid culture expansion, an increase inyields of 100-1000 fold, and reduced cost for production of L.intracellularis of European origin. As a result, the abundant supply ofL. intracellularis bacteria produced is readily attenuated for vaccineproduction purposes. The method of growing L. intracellularis insuspension greatly increases the ease, speed, and number of bacteriumavailable for this purpose. The more cells and cell divisions whichoccur, the greater the level of mutations occurring which areadvantageous in vaccine development. Thus, growth in suspensionsincreases the expression of important immunogens controlled byenvironmentally regulated genes and their expression products.

The resulting attenuated isolates can be cultivated in tissue culturemonolayers but are preferably cultivated in suspension cultures. Othermeans of attenuation can include chemical attenuation by the use of, forexample, N-methyl nitrosoguanidine and others known in the art. Whetherby multiple passage or chemical means, an attenuated L. intracellularisis produced and selected for vaccine preparation. In a preferredembodiment, the resulting attenuated isolate is ATCC accession No.PTA-4926.

The vaccine antigen can be harvested by centrifugation ormicrofiltration as described above. The antigen is then standardized ata defined level based on the optimum host animal immune response,determined by a dose titration in the host animal species. The bacteriamay be inactivated by methods known in the art, e.g., by using 0.3%formalin or other inactivating agents to prepare a killed vaccine. Theantigen is then incorporated into a suitable adjuvant, such as aluminumhydroxide or mineral oil to enhance the immune response. The antigen isthen used to vaccinate the host via intramuscular or subcutaneousinfection, in the case of swine at about 3-4 weeks of age, with abooster dose if necessary.

Preferably, the bacteria is serially passaged to induce and select foran attenuated, avirulent live culture. The culture is tested in the hostanimal for signs of attenuation. The culture is harvested as describedearlier and lyophilized. Swine, for example, are orally vaccinated with1×10⁴ to 1×10⁶ bacteria. About twenty-eight days after vaccination, theswine are orally inoculated with about 1×10⁷ organisms from a lesspassaged (less than 30 passages in vitro past the original isolationfrom the intestinal homogenate) virulent culture of L. intracellularis.Infected animals are necropsied 21 days after challenge and the smallintestines observed for gross lesions as well as microscopic lesions.PCR, indirect fluorescent antibody (IFA) or immunohistochemistry (IHC)should also be performed.

About eighty percent of the control animals will show gross ormicroscopic lesions and test positive for the presence of L.intracellularis in the mucosal cells of the intestines using either PCR,IFA or IHC testing methods. Vaccinated animals will have normal mucosalsurfaces as determined by histological observations and will be negativeby PCR testing 3 to 4 weeks post inoculation.

Generally, an attenuated immunogenic L. intracellularis isolate isproduced after continuous culture for about 150 days to about 250 days,during which time the culture is passaged about 50-100 times. However,one skilled in the art knows that other attenuated cultures may beproduced by varying these figures.

The vaccine product of the invention can be lyophilized. Afterharvesting, the isolate can be concentrated by various methods known inthe art and can be mixed with a stabilizer, e.g. sucrose gelatinstabilizer. The vaccine product can then be subjected to freezing anddrying (lyophilization). Generally, the freezing step comprises rampingto about −45° C.±3° C. and holding for about 150 minutes to about 480minutes. The drying step can comprise primary and secondary dryingsteps. For example, the primary drying step can comprise: (a) ramping tobetween about −30° C. to about −5° C. and holding for between about 120minutes to about 1000 minutes, and, optionally (b) ramping to betweenabout −5° C. to about 5° C. and holding for between about 150 minutes toabout 2000 minutes. The secondary step generally comprises ramping toabout 27° C.±5° C. and holding for between about 330 minutes to about1120 minutes. One skilled in the art knows that these ranges can beadjusted depending on conditions, e.g., starting volume.

A vaccine is then prepared comprising an immunologically effectiveamount of the attenuated L. intracellularis in a pharmaceuticallyacceptable carrier. In a preferred embodiment, a vaccine comprises ATCCaccession No. PTA-4926 in a pharmaceutically acceptable carrier. Thecombined immunogen and carrier may be an aqueous solution, emulsion orsuspension. An immunologically effective amount is determinable by meansknown in the art without undue experimentation given the teachingscontained herein. In general, the quantity of immunogen will be between5 and 5000 micrograms, and between 10^(2.0) and 10^(9.0) TCID₅₀,preferably between 10^(3.0) and 10^(6.0) TCID₅₀, more preferably between10^(4.0) and 10^(5.0) TCID₅₀, when purified bacteria are used.

The present invention also encompasses combination vaccines comprisingthe attenuated L. intracellularis isolate designated ATCC accession No.PTA-4926 and antigenic material from at least one other pathogen,including but not limited to: Salmonella spp. (e.g., Salmonellacholeraesuis, Salmonella typhimurium), Erysipelothrix spp. (e.g.,Erysipelothrix rhusiopathiae), Haemophilus spp. (e.g., Haemophilusparasuis), Mycoplasma spp. (e.g., Mycoplasma hyopneumonia), Leptospiraspp., Clostridium spp. (e.g., Clostridium perfingens, Clostridiumdifficile), Streptococcus spp. (e.g., Streptococcus suis), Brachyspiraspp. (e.g., Brachyspira hyodysenteriae), Bordetella (e.g., Bordetellabronchiseptica), Pasteurella spp. (e.g., Pasteurella multocida),circovirus (e.g., porcine circovirus type 2), porcine reproductive andrespiratory syndrome (PRRS) virus, swine influenza virus (SIV),coronovirus (e.g., transmissible gastro-enteritis (TGE) virus, porcinerespiratory corona virus), parvovirus, or Escherichia coli; and apharmaceutically acceptable carrier.

In one embodiment, the combination vaccine comprises the attenuated L.intracellularis isolate designated ATCC accession No. PTA-4926 andantigenic material from Salmonella choleraesuis, Erysipelothrix spp.Clostridium spp, Brachyspira spp., transmissible gastro-enteritis (TGE)virus, and Escherichia coli; and a pharmaceutically acceptable carrier.Antigenic material from Clostridium spp. can include, but is not limitedto, Clostridium perfingens and Clostridium difficil. Antigenic materialfrom Erysipelothrix spp. can include, but is not limited to,Erysipelothrix rhusiopathiae.

In another embodiment, the combination vaccine comprises the attenuatedL. intracellularis isolate designated ATCC accession No. PTA-4926 andantigenic material from Salmonella choleraesuis and Erysipelothrix spp.;and a pharmaceutically acceptable carrier. In another embodiment, thecombination vaccine comprises the attenuated L. intracellularis isolatedesignated ATCC accession No. PTA-4926 and antigenic material fromSalmonella choleraesuis and Erysipelothrix rhusiopathiae; and apharmaceutically acceptable carrier.

In another embodiment, the combination vaccine comprises the attenuatedL. intracellularis isolate designated ATCC accession No. PTA-4926 andantigenic material from at least one other pathogen, including but notlimited to: Clostridium spp. e.g., Clostridium tetani), equine influenzavirus (EIV) (e.g., EIV-1, EIV-2), equine herpes virus (EHV) (e.g.,EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, EHV-6, EHV-7,), alphavirus (e.g.,eastern encephalitis virus, western encephalitis virus, Venezuelanencephalitis virus), or West Nile virus.; and a pharmaceuticallyacceptable carrier.

The vaccines according to the invention are generally administered tosusceptible animals, preferably swine, in one or more doses. The live orkilled vaccine may be administered 1 or 2 times at 2 week intervals. Forthe attenuated, live vaccines, one dose is preferred. The preferredroutes of administration of attenuated live isolates are intramuscular,oral or intranasal, but intramuscular and subcutaneous injection routesare most preferred for the killed vaccine.

Effective diagnosis of PPE has also been hindered by the time requiredto culture the causative bacteria. As a result of the present invention,development of diagnostic tools promoting rapid and accurate assays forthe presence of L. intracellularis in biological samples taken fromswine and other animals susceptible to PPE is now possible.

The L. intracellularis bacteria of European origin of the instantinvention, or components derived from such bacteria, can be used as anantigen in an ELISA or other immunoassay, such as an immunofluorescentantibody test (“IFA”), to detect antibodies to L. intracellularis in theserum and other body fluids of animals suspected of being infected withthe bacteria. The presently preferred immunoassay is an IFA as describedin the example below. Alternatively, the bacteria of the instantinvention can be used in a Western Blot assay.

The preferred ELISA protocol according to the invention is as follows:

-   -   1. Add 0.1 ml/well antigen diluted in coating buffer. Incubate        for 18 hours at 4° C.    -   2. Wash 3 times with PBS.    -   3. Add 0.25 ml of blocking buffer to each well of plate.        Incubate 1 to 2 hours at 37° C.    -   4. Wash 3 times with wash buffer.    -   5. Dilute serum in blocking buffer and add 0.1 ml to the first        wells of plate. Make serial 1:2 dilutions across the plate.        Incubate for 1 hour at 37.    -   6. Wash 3 to 5 times with wash buffer.    -   7. Dilute conjugate in blocking buffer and add 0.1 ml to wells        of plate and incubate for 1 hour at 37° C.    -   8. Wash 3 to 5 times with wash buffer.    -   9. Add substrate.    -   10. Measure absorbance of light with a spectrophotometer.    -   11. Wells in which antigen was not added are used as blanks.    -   12. Positive and negative control swine serum should also be        used with each test.

The preferred Western Blot protocol is as follows:

-   -   1. Run antigen on 12% SDS-PAGE and transfer to nitrocellulose        membrane.    -   2. Place membrane in blocking buffer for 2 hours.    -   3. Remove blocking buffer and rinse with PBS for 1 minute.    -   4. Dilute serum in blocking buffer and add to membrane. Incubate        for 2 hours at room temperature.    -   5. Wash 3 times with wash buffer (5 minutes for each wash).    -   6. Dilute conjugate in blocking buffer and add to membrane.        Incubate for 1 hour at room temperature.    -   7. Wash 3 times with wash buffer.    -   8. Add substrate for 10 minutes or until strong banding occurs.    -   9. Rinse with PBS.    -   10. Air dry and store in the dark.

The L. intracellularis bacteria of European origin of the instantinvention, or components derived from such bacteria, can also be used toprepare antiserum or antibodies for diagnostic, prophylactic, ortherapeutic use. The L. intracellularis bacteria of European origin ofthe instant invention, or components derived from such bacteria, can beadministered to a non-human animal in an amount effective to elicit animmune response and the antiserum or plasma containing antibodies to theL. intracellularis bacteria, or components derived from such bacteria,can be collected according to methods known in the art and describedherein.

The present invention is further described in the following exampleswhich are provided for illustrative purposes only and are not to beconstrued as limiting. Indeed, other variants of the invention will bereadily apparent to one of ordinary skill in the art.

All publications and patents cited herein are incorporated by referencein their entireties.

EXAMPLE 1 Production of L. Intracellularis Vaccine

Isolation and Attenuation of L. intracellularis from the Intestines ofEuropean Pigs with Porcine Proliferative Enteropathy (PPE):

L. intracellularis virulent isolate DK 15540 (DK 15540, DK-15540 and15540 are used interchangeably herein) was isolated by the University ofMinnesota from an ileal homogenate of a Danish pig infected with acuteporcine hemorrhagic enteropathy. This isolate has been deposited underthe Budapest Treaty with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209 on Jan. 9, 2003 andassigned accession number PTA-4927. The isolation process includedscraping the mucosa from the ileum, homogenizing, trypsinizing for 30minutes, and passing through a tissue grinder. The ileal homogenate wasthen passed through a series of filters consisting of 5.0, 1.0, and 0.65μm. The homogenate was diluted in sucrose phosphate glutamate bufferwith 10% fetal bovine serum (FBS). Aliquots (6×1 ml) of homogenate weremade and stored at less than −70° C. The homogenate was used as inoculumto infect T-75 cm² flasks of McCoy cells. Cultures were monitored dailyfor McCoy cell infection by scraping McCoy cell monolayers, lysing cellsby potassium chloride treatment, and placing the concentrated cellpellet on microscope slides stained by IFA using monoclonal antibodiesspecific for L. intracellularis. After eleven passages on anchoragedependent cell cultures, inoculum from passage eleven was transferredinto a 250 ml spinner flask containing McCoy cells and grown insuspension until harvest. The Danish isolate of L. intracellularis (ATCCaccession No. PTA-4927) was attenuated by continuous in vitro passage inMcCoy cells for 80 weeks and tested for identity by monoclonalantibodies. The attenuated isolate was designated B3903 (B3903, B 3903and B-3903 are used interchangeably herein). Isolate B3903 was depositedunder the Budapest Treaty with the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209 on Jan. 9, 2003 andassigned ATCC accession No. PTA-4926.

Cultures:

Identification

Characteristic growth requirements, PCR reactions, and monoclonalantibody reactions were used to identify L. intracellularis Master andWorking Seed materials.

Purity

Purity of Master Seed and Working Seed of L. intracellularis wasdetermined by examining cultures with monoclonal antibody stains,conventional extraneous agents tests for bacteria and viruses and bysterility mycoplasma tests.

Virulence

Master Seeds of L. intracellularis were not virulent as demonstrated bythe lack of ability of the Master Seed and Back passage inoculum toproduce clinical signs and gross lesions that are observed insusceptible swine following exposure of virulent L. intracellularis, asfurther illustrated in Example 2, infra.

Range of Subcultures

Final harvested material from production of L. intracellularis did notexceed eleven passages from the Master Seed.

Medium Composition

EU McCoy Master Cell Stock (MCS) were grown and maintained in Dulbecco'sModified Eagle Medium with Ham's Fortified F12 (DMEM/F12) and 1-10%(v/v) newborn bovine serum (NBS) or fetal bovine serum (FBS) (cellgrowth and maintenance media).

Master and Working Seed were stored in DMEM/F12 with 1-10% (v/v) NBS orFBS and 5-15% (v/v) glycerol (master and working seed storage media).

The final harvest product was stored in sucrose gelatin stabilizer (SGS)(final product storage media).

Propagation

Master and Working Seed cultures of L. intracellularis were propagatedin EU McCoy MCS using cell growth and maintenance media as describedsupra and stored at less than or about −35° C.

Source of Tissue

L. intracellularis seed and production organisms were grown in McCoyMaster Cell Stock (ATCC accession number CRL 1696, batch numberF-10422). The mast cell stock was identified as 3894MMCSS at passage X.The master cell stock was passaged an additional six times andidentified as EU McCoy MCS X+0. EU McCoy MCS passage X+0 was stored at−70° C.±5° C. or colder. Production vaccine was made in EU McCoy MCScell line subcultures through the 40^(th) passage.

Culture Containers

EU McCoy MCS were propagated in tissue culture flasks with 25-150 cm²surface area, Costar cell cubes, in roller bottles with 850 cm² to 2,2250 cm², in spinner flasks up to 40 L capacity, and in bioreactorshaving 3 L to 500 L capacity.

Seed cultures of L. intracellularis were grown in 250-40,000 ml spinnerflasks, 850 cm² to 2,250 cm² roller bottles, tissue culture flasks with25-150 cm² surface area, or in bioreactors having 3 L to 500 L capacity.

Production cultures of L. intracellularis were grown in 6 L to 40 Lspinner flasks or in bioreactors having 3 L to 500 L capacity.

Methods of Preparing Suspensions for Seeding or Inoculation

Seeding Cultures

Frozen or fresh L. intracellularis Master or expanded Working Seeds werethawed at room temperature (25° C.±3° C.) or at 37° C.±2° C.Bioreactors, spinner flasks or bottles previously seeded with McCoycells at a cell density of 50,000 to 500,000 cells/ml were infected withL. intracellularis at a concentration of 1 to 10% (v/v) or MOI of 0.08to 1.0. The infected culture was incubated under reduced oxygenconcentrations by overlaying with a gas mixture composed of 86% N₂, 4%H₂ and 10% CO₂. The culture was incubated for 3 to 10 days at 37° C.±2°C., pH 6.7 to 7.3, and with continuous agitation (10 to 100 rpm) tomaintain adequate mixing for the cells to remain in suspension.

Production Cultures

Frozen or fresh L. intracellularis Master or expanded Working Seeds werethawed at room temperature (25° C.±3° C.) or at 37° C.±2° C. Bioreactorsor spinner flasks 3 L to 500 L capacity) previously seed (0 to 7 days)with McCoy cells at a cell density of 50,000 to 500,000 cells/ml wereinfected with L. intracellularis at a concentration of 1 to 10% (v/v) orMOI of 0.08 to 1.0. The infected culture was incubated under reducedoxygen concentrations by overlaying or sparging with a gas mixturecomposed of 96% N₂, 4% H₂.

The culture was incubated for 3 to 8 days at 37° C.±2° C., pH 6.7 to7.3, and with continuous agitation (10 to 100 rpm) to maintain adequatemixing for the cells to remain in suspension.

Inoculation Techniques for Seed and Production Cultures

Seed Cultures

Up to 10% (v/v) of Master or Working Seed are inoculated (MOI=0.08 to1.0) into bioreactors, spinner flasks or bottles seed with McCoy cellsat 0-7 days in growth medium.

Production Cultures

Up to 10% (v/v) of Production Seed was inoculated (MOI=0.08 to 1.0) intoan appropriate volume of growth medium seeded with McCoy cells at 0 to 7days with the appropriate McCoy cell density in 3 L to 500 L capacityvessels.

Incubation of Microorganisms

The cultures were incubated at 37° C.±2° C. for 3 to 10 days in areduced oxygen atmosphere with agitation to maintain the suspension.Additional medium and/or McCoy cells can be added to continue the growthprocess.

Cultures were observed macroscopically during the incubation period forevidence of abnormal growth or signs of contamination.

Harvest:

Handling and Preparation of Cultures

Cultures were examined for signs of adequate bacterial growth byindirect fluorescent antibody (IFA) staining. Cultures that were readyfor harvest exemplified 60 to 100% cell infectivity. Percent infectivitywas determined by observation of at least three fields, each fieldcontaining enough McCoy cells to fill at least 80% of the area. To beconsidered infected, approximately 50% of the cell is filled withbacteria.

Potency of the harvest culture is tested by titration of the sample onMcCoy cells which are fixed and stained using specific monoclonalantibody (anti-L. intracellularis monoclonal antibody VPM 53 Lot 31599or equivalent; anti-mouse IgG-fluorescein conjugate (FITC) (ICN No.55499) after 6 days of incubation at 37° C.±2° C.

Cultures were examined visually for any obvious signs of contamination.Harvest occurred 3 to 10 days post inoculation.

Harvesting Techniques and Specifications

Cell and fluid contents in the production culture bioreactor, spinnerflasks and bottle were partially or fully collected in a sterilereceiving vessel. Each production culture bioreactor, spinner flask andbottle was harvested individually or the contents of several vesselswere pooled with the addition of SGS and stored at 1° C. to 7° C. orcolder. Harvested production culture was sampled for potency by TCID₅₀and identification by IFA staining.

Production cultures exhibited at least 4.9 TCID₅₀/ml by IFA staining andwere free of any evidence of contamination upon microscopic observation.

Preparation of Vaccine Product:

Concentration Methods

The vaccine product can be concentrated by various methods, e.g., byallowing the culture to settle with subsequent decanting of thesupernatant, by membrane filtration (0.22 μm or smaller), perfusion, orby centrifugation.

Sucrose Gelatin Stabilizer (SGS)

Hydrolyzed gelatin solution is prepared by mixing gelatin with deionizedwater or water for injection at approximately 25% final total volume ofSGS batch size and hydrolyzing in an autoclave for 120 minutes at 121°C.

The hydrolyzed gelatin solution (40.0 g/L) was then mixed with deionizedwater or water for injection at approximately 75% final total volume ofSGS batch size. Potassium hydroxide (AR) (0.548 g/L), L-glutamic acid(1.440 g/L), dipotassium phosphate (AR) (2.508 g/L), potassiumdihydrogen phosphate (AR) (1.030 g/L), and sucrose (AR) (150.00 g/L)were added and the solution was thoroughly mixed. The pH of thestabilizer was then adjusted to 6.8 to 7.0 with hydrochloric acid orsodium hydroxide solutions. Deionized water or water for injection wasadded to 100% of desired final volume of the SGS. The completestabilizer was thoroughly mixed, and the entire solution was sterilizedby filtration through a 0.1 micron filter.

Example of Assembly of Units to Make a Serial is Shown in Table 1: TABLE1 L. intracellularis 200,000-300,000 ml sucrose gelatin stabilizer (SGS)100,000 ml (25% v/v) DMEM/F12 (May be added to 0-150,000 ml standardizethe product) TOTAL VOLUME 400,000 mlVolume of an average serial was 50 L to 500 L.

Lyophilization

The vaccine product was lyophilized according to the procedure outlinedin Table 2 for a 10 dose cycle (6.0 ml fill) or Table 3 for a 50/100dose cycle (10.0 ml fill). TABLE 2 Rate Hold Pressure STEPS ° C.(minutes) (minutes) (mT) PRE-COOL* 5° Na Na Atm FREEZE −47° ± 3° As fastas 150 Atm possible 1° DRYING, 1st −15° ± 2° 120 120 100-150 1° DRYING,2nd  0° ± 2° 120 180 100-150 2° DRYING, 1^(st)  32° ± 2° 240 180 60-802° DRYING, 2nd  26° ± 2° 240 As fast as 60-80 possibleTotal Time: 1352 minutes (22.5 hrs)*Shelves are pre-cooled to 5° ± 2° C. during the loading of thelyophilizer.

TABLE 3 Rate Hold Pressure STEPS ° C. (minutes) (minutes) (mT) PRE-COOL*5° N/A N/A Atm FREEZE −48° ± 3° 60  90 Atm 1° DRYING −15° ± 2° 60 1500100-150 2° DRYING  26° ± 2° 60  600 60-80Total Time: 2370 minutes (39.5 hrs)*Shelves are pre-cooled to 5° ± 2° C. during the loading of thelyophilizer.

EXAMPLE 2 Safety of L. Intracellularis Vaccine

Purpose:

The objectives of this study were two-fold. The first objective of thisstudy was to observe and compare the incidence of disease caused bythree different low passage isolates (two of U.S. origin and one ofEuropean origin) of L. intracellularis in pigs at 6½ weeks of age. Thesecond objective was to observe the safety of two high passage isolates(both of European origin) of L. intracellularis in pigs at 6½ weeks ofage.

Materials and Methods:

Test Substances

-   -   1. L. intracellularis low passage U.S. isolate N343    -   2. L. intracellularis low passage U.S. isolate N101494.    -   3. L. intracellularis low passage EU isolate DK 15540 p20    -   4. L. intracellularis high passage EU isolate DK 15540 p60    -   5. L. intracellularis high passage EU isolate DK 15540 p80        (Master Seed designation B3903).

Formulation of Test Substances

Low passage isolates were grown continuously for 10-20 weeks afterisolation in McCoy cell suspension. High passage isolates were growncontinuously for 60-80 weeks after isolation in McCoy cell suspension.All cultures were harvested via centrifugation at 10,000 RPM for 15minutes. The McCoy cell culture pellets containing Lawsonia wereresuspended in Sucrose-Phosphate-Glutamine (SPG) solution with 10% FBS.

Storage of Test Substances

Harvested cultures were stored at −70° C. until the day of challenge.Challenge cultures of the same isolate but from various harvest dateswere thawed and combined into plastic vaccine bottles, labeled, andstored at 4° C. or on ice until the time of challenge.

Assay of Test Substances

TCID₅₀ was performed on all pooled challenge isolates at the time ofchallenge (day 0). The average titers (n=3) (TCID₅₀/ml) were as followsin Table 4: TABLE 4 Test Substance Average Titer (TCID₅₀/ml) L.intracellularis N343 6.4 L. intracellularis N101494 6.1 L.intracellularis DK 15540 p20 6.2 L. intracellularis DK 15540 p60 6.87 L.intracellularis DK 15540 p80 7.4

Study Design

The study consisted of five experimental groups and one control group.On day 0 of the study, Group 1 (10 pigs, 6½ weeks of age) received one10 ml or equivalent intragastric (IG) dose of L. intracellularis lowpassage U.S. isolate N343. Group 2 (10 pigs, 6½ weeks of age) receivedone 10 ml or equivalent IG dose of L. intracellularis low passage U.S.isolate N101494. Group 3 (10 pigs, 6½ weeks of age) received one 10 mlor equivalent IG dose of L. intracellularis low passage EU isolateDK15540 p20. Group 4 (10 pigs, 6½ weeks of age) received one 10 ml orequivalent IG dose of L. intracellularis high passage EU isolate DK1554060 week. Group 5 (20 pigs, 6 weeks of age) received one 10 ml orequivalent dose of L. intracellularis high passage EU isolate DK 15540p80. Group 6 (10 pigs, 6½ weeks of age) designated as “Strict Controls”did not receive a treatment.

Daily health observations were made from initiation of study to the dayof challenge of appropriate test animals. Clinical health (behavior,appetite, body condition, hair coat, and stool consistency on a scale of1 to 4 were scored daily from day of challenge (day 0) to termination ofstudy (day 21). Average daily weight gains (ADWG) were calculated fromday of challenge (day 0) to termination of study (day 21). Fecalshedding of L. intracellularis was evaluated on days 0, 7, 14, and 21.The one animal that died (from Group 1) throughout the study wasexamined for gross and microscopic lesions. Death was determined to bedue to lesions associated with PPE confirmed by histology and PCRanalysis; the animal was not replaced. Qualitative analysis of Lawsoniacontent in feces was evaluated by PCR along with histological evaluationfor L. intracellularis on the ileum and colon. Serum was collected ondays 0, 7, 14, and 21 of the study.

Results:

Summary of Study Results TABLE 5 No. of Titer Fecal Gross Clinical GroupTreatment Pigs (TCID₅₀/ml) Serology Shedding PCR FA Histology LesionsADWG Scores 1 N343 9 7.4 19% 11% 0% 11% 44% 1 0.61 5.23 2 N101494 10 7.110% 10% 10% 30% 30% 1.2 0.8 5.15 3 DK15540p20 10 7.2 18% 13% 30% 10% 20%1.2 0.86 5.01 4 DK15540p60 10 7.87 5% 0% 0% 0% 10% 1 0.9 5 5 DK15540p8020 8.4 0% 0% 0% 0% 0% 1 1.05 5 6 Strict 10 0 0% 0% 0% 0% 0% 1.05 0.91 5Controls

General Observations

Daily health observations were made until challenge. The clinicalcondition of the animals was evaluated daily following challenge for theduration of the study. Observations included: behavior, appetite, bodycondition, hair coat, and stool consistency. The clinical conditions ofthese animals were evaluated based on a numerical point system, whichreflects the severity of illness. Scores ranged from 1 to 4 for eachparameter. A score of 1 was given to an animal with a normal, healthyappearance, a score of 3 for an animal demonstrating severe clinicalsigns, and a score of 4 for an animal that has deceased. The averagedaily score for the strict controls, DK15540 p60, and DK15540 p80 was5.0. The average daily scores for low passage material was N343 (5.23),N101494 (5.15), and DK15540 p20 (5.01). Statistical analysis on theseresults indicated no differences among treatment and control groupsusing Kruskal-Wallis Rank Sum Test.

Average Daily Weight Gains (ADWG)

Average daily weight gains were calculated from the time of challenge(day 0) to the termination of the study (day 21). The average weightgain per day for the strict control group was 0.9 pounds. The averageweight gain for the low passage treatment groups was only 0.6 (N343),0.8 (N101494), and 0.86 (DK15540 p20) pounds/day. The high passagetreatment groups revealed the same or increased average weight gain perday compared to the strict control group that didn't receive challengewith 0.9 pounds/day (DK15540 p60) and 1.05 pounds/day (DK15540 p80)respectively. The mean difference in average daily weight gains wassignificantly lower in the N343 treatment group compared to higherpassage treatment groups (DK15540 p60 and p80) and strict control groupat day 21 of the study. (Pearson Chi-square p<0.05).

Seroconversion

Seroconversion to Lawsonia exposure in pigs was measured by testing forthe presence of anti-Lawsonia antibodies using an IFAT assay. On day 0,only the N343 treatment group observed detectable seroconversion in 2/10pigs. Day 7 observed 2/9 pigs (N343) and 1/10 pigs (DK15540 p20) IFApositive for Lawsonia antibodies. On day 14, 2/9 pigs were IFA positivein N343 treatment group, 1/10 in DK15540 p20 and p60 respectively. Day21 revealed 1/10 pigs (N343), 4/10 pigs (N101494) and 6/10 pigs (DK15540p20) were IFA positive while high passage treatment groups (DK15540 p60and p80) show no detectable seroconversion. Seroconversion to Lawsoniaexposure increased in treatment groups receiving low passage L.intracellularis of both the U.S. and EU isolates day 21 of the study.

Fecal Shedding

PCR testing of the feces demonstrated shedding of L. intracellularisbeginning on day 14 where 4/9 in the N343, 4/10 in the N101494, and 5/10in the DK15540 p20 low passage animals tested positive. Both highpassage treatment and strict control groups were PCR negative for day14. On day 21, DK15540 p20 treatment group had 1 animal PCR positivewhile all other treatment and control groups were PCR negative. Noevidence of shedding was observed in high passage isolate groups(DK15540 p60 and p80) using PCR throughout the study. PCR positiveanimals indicating active shedding of Lawsonia in their feces were moresignificant in the low passage isolate groups (N343, N101494, andDK15540 p20) on day 14 of the study than high passage isolate groups andstrict controls (Pearson Chi-square p<0.05).

PCR at Day 21: Ileums and Colons

PCR testing of mucosal scrapings from ileums and colons was performedafter necropsy (day 21). Samples that were PCR positive for L.intracellularis colonization were (2/10 colons) in N101494 and (2/10ileums, and 4/10 colons) in DK 15540 p20 low passage isolate groups. Allother treatment and control groups were PCR negative in tissues day 21of the study. Results indicated ileums and colons of pigs in DK15540 p20were significantly more colonized with L. intracellularis compared toall treatment and control groups (*Pearson Chi-square p<0.05).

Histology

Sections of the terminal ileum and colon were collected at necropsy (day21) and placed in buffered formalin for histological analysis. Presenceof intracellular bacteria and crypt hyperplasia was observed in tissuesstained with Hematoxylin and Eosin (H&E) and Warthin-Starry silverreagents of 4/9 pigs (N343), 3/10 pigs (N101494), 2/10 pigs (DK15540p60) and 1/20 pigs (DK15540 p80). Lesion development was confirmed byflorescent antibody staining using monoclonal antibodies againstLawsonia intracellularis in 1/9 pigs (N343), 3/10 pigs (N101494), and1/10 pigs (DK15540 p60). FA detected no lesions caused by Lawsonia inthe colons of all treatment and control groups. FA results indicatedsignificant lesion development in ileums of pigs in N101494 treatmentgroup compared to all treatment and control groups. H&E/silver stainingshowed significant lesion development associated with PPE in N343treatment group compared to all treatment and control groups. (PearsonChi-square p<0.05).

Gross Scores

Ileums and colons were scored at the time of necropsy (day 21) forlesions associated with PPE. Tissues were given a score of 1 for normalappearance (no lesion development), a score of 2 for lesionsdemonstrating mild thickening, 3 for moderate thickening, and a score of4 for severe thickening. Strict controls had an average clinical scoreof 1.05, N343 (1.0), N101494 (1.2), DK15540 p20 (1.2), and DK15540 p60and p80 (1.0). Average gross lesion scores indicated no statisticaldifference between control and treatment groups using ANOVA test formultiple comparisons.

Conclusions:

Based on the data collected, this study demonstrated that pigschallenged with a low passage dose of N343, N101494, and DK15540 with aTCID₅₀ greater than 1×10⁷ bacteria/dose increases the incidence of PPEin these animals. High passage isolates (DK15540 p60 and p80) given topigs of the same age with a TCID₅₀ greater than 1×10⁷ were proven safeand show reduction of colonization and lesion development associatedwith PPE. This conclusion was based on PCR on the mucosa of the ileum,histopathology, and FA stains of tissue sections.

A reduction of shedding L. intracellularis in the feces determined byPCR was evident in high passage isolates compared to low passageisolates. Average daily weight gains calculated for all treatment andcontrol groups support this conclusion by demonstrating positive uniformdaily weight gain in groups given the high passage isolate and strictcontrols compared to groups given low passage isolates especiallyanimals in the N343 treatment group. This observation indicatesreduction of weight gains in animals given low passage material whichsupports adequate and similar grow performance of animals given highpassage material with animals receiving no challenge material. Comparedto the strict controls, the high passage isolates showed no negativeimpact on weight gain and overall health based on clinical scores.

EXAMPLE 3 Efficacy and Minimal Protective Titer

Purpose:

The objectives of this study were to determine the minimal protectivetiter of a vaccine comprising isolate B3903 (lyophilized) (DK 15540,passage 80) (“B3903 (Lyophilized) vaccine” and “B3903 vaccine” are usedinterchangeably herein) administered by oral drench in pigs 3 weeks ofage and to demonstrate efficacy against a virulent heterologous pureculture challenge with low passage L. intracellularis, the causativeagent of Porcine Proliferative Enteropathy (PPE) in swine.

Materials and Methods:

Test Substance: Attenuated Live Culture of L. intracellularis, IsolateB3903

Formulation of B3903 (Lyophilized) Vaccine

DK 15540 isolates were grown continuously for 80 weeks after isolationin McCoy cell suspension. All cultures were harvested via centrifugationat 10,000 RPM for 15 minutes. The McCoy cell culture pellets containingLawsonia were resuspended in Sucrose-Phosphate-Glutamine (SPG) solutionwith 10% FBS. Desiccation was performed as described in Example 1,supra. The lyophilized product is reconstituted in water, q.s. ad 2.0ml, for injections.

Storage of B3903 (Lyophilized) Vaccine

The vaccine was stored at 2° C.-8° C. until ready for use. Afterresuspension, the vaccine was stored on ice until administration.

Doses of B3903 (Lyophilized) Vaccine

-   -   1. High dose (Treatment Group 1): 1×2 mL (6.0 logs/dose) via        direct oral drench on day 0 of the study.    -   2. Medium dose (Treatment Group 2): 1×2 mL (4.9 logs/dose) via        direct oral drench on day 0 of the study.    -   3. Low dose (Treatment Group 3): 1×2 mL (3.8 logs/dose) via        direct oral drench on day 0 of the study.

Test Substance: Placebo

A placebo consisting of uninfected McCoy tissue culture cells suspendedin DMEM/F12 growth medium fortified with 5% NBS was given to treatmentGroups 4 (challenge control) and 5 (negative control) on day 0 of thestudy. This substance was administered to piglets in treatment Group 4by direct oral drench and given 1×2 mL of placebo per test animal.

Test Substance: L. intracellularis N101494 Virulent Challenge

L. intracellularis N101494 was obtained from the intestines of a 12 weekold pig from an Indiana farm (U.S. Pat. No. 5,714,375).

Formulation of L. intracellularis N101494 Virulent Challenge

L. intracellularis challenge material was grown continuously in McCoycell suspension no more than 30 passes after initial isolation frominfected gut tissue. Active cultures (2×3 L) identified as SF 1422 andSF 1423 in addition to (1 L) SF 1421 were grown in McCoy cell suspensionfor 7 days to 15-30% McCoy cell infection. On the day of challenge (day21), active cultures were harvested via centrifugation at 10,000 RPM for15 minutes and cell pellets resuspended in 350 mls total volume with SPGstabilizer. Harvested active culture was pooled with 300 mLs of frozen10× to 20× concentrated challenge stocks of low passage N101494 atvarious passages (pass 24 to 27 post isolation).

Storage of L. intracellularis N101494 Virulent Challenge

Final formulations ready for challenge were stored at 2° C. to 8° C. oron ice until inoculation.

Pre/Post-Vaccination and Challenge Titers

Results from the TCID₅₀ assay verified the amount of live L.intracellularis administered to each test animal per dose duringvaccination and challenge. The average titers (n=5) of pre and posttitrations for the B3903 vaccine and the challenge material (L.intracellularis N101494) were as follows in Table 6: TABLE 6 AverageAverage Total logs/dose (2 ml) logs/dose (2 ml) Average TreatmentPre-vaccination Post-vaccination logs/ Group (logs/dose) (TCID₅₀/ml)(TCID₅₀/ml) dose 1 B3903 vaccine 6.07 5.89 6.0 high dose 2 B3903 vaccine4.94 4.84 4.9 medium dose 3 B3903 vaccine 3.9 3.5 3.8 low dose 4Challenge 7.85 7.57 7.71 Controls

Study Design

Sixty-five healthy L. intracellularis negative weaned piglets at 3 weeksof age were randomly divided into 5 treatment groups and housedseparately throughout the study. On day 0, treatment Group 1 (15 pigs)received a 2 mL dose (6.0 logs/dose) of B3903 vaccine by direct oraldrench. Treatment Group 2 (15 pigs) received 1×2 mL dose of B3903vaccine titrated at 4.9 logs/dose by direct oral drench. Treatment Group3 (15 pigs) received 1×2 mL dose of B3903 vaccine titrated at 3.8logs/dose by direct oral drench.

Treatment Groups 4 and 5 (10 pigs/group) received 1×2 mL dose of placeboby direct oral drench.

On day 21 of the study (3 weeks post vaccination), test pigs intreatment Groups 1-4 received 1×10 mL dose of virulent low passage pureculture L. intracellularis heterologous isolate N101494 by gastricgavage.

On day 42 of the study (3 weeks post challenge), all treatment Groups(1-5) were euthanized and necropsied for gross and microscopic lesionanalysis for PPE.

Daily health observations were made from study initiation to the day ofchallenge for each test animal. Clinical health (diarrhea, behavior, andbody condition) were scored daily from day of challenge (day 21) totermination of study (day 42). Weights were taken on day of vaccination(day 0), day of challenge (day 21) and on day of study termination (day42) to calculate average daily weight gains of each treatment group.Fecal shedding of L. intracellularis was evaluated by polymerase chainreaction (PCR) by testing fecal swabs (f-PCR) on days 0, 7, 14, 21, 28,35, and 42 of the study. All animals euthanized at study termination(day 42) were examined for gross and microscopic lesions. Qualitativeanalysis of bacterial content in tissues was evaluated by PCR (t-PCR)along with histological evaluation for L. intracellularis in the ileum,colon, tonsil, and mesenteric lymph node day 42 of the study. Serum wascollected on days 0, 7, 14, 21, 28, 35, and 42 of the study. Serum wastested using the indirect florescent antibody test (IFAT) to detectanti-Lawsonia antibodies in test animals. Treatment group comparisonswere made by data analysis of average daily weight gains postvaccination and post challenge, clinical scores, seroconversion rates(IFAT), colonization (t-PCR), fecal shedding (f-PCR), gross lesion, andmicroscopic lesion development by immunohistochemistry (IHC).

Results:

Summary of Primary Results TABLE 7 Average Average Average Average GrossGross Micro- Micro- Treat- Pigs Treatment. Lesion Lesion Lesion Lesionment per Group ID Scores Scores scores Scores Group Group (logs/dose)(ileum) (colon) (ileum) (colon) 1 15 B3903 vaccine 1.5 ab 1.2 a 0.2 ab0.1 a high dose (6.0) 2 15 B3903 vaccine 1.2 ab 1.0 a 0.4 ab 0.0 amedium dose (4.9) 3 15 B3903 vaccine 2.5 b 1.5 b 1.0 b 0.3 a low dose(3.8) 4 10 Challenge 3.6 c 2.2 b 2.4 c 1.5 b Controls 5 10 StrictControls 1.0 a 1.0 a 0.0 a 0.0 a*Like letters indicate no significant difference (p < 0.05)

Final test results for each treatment group revealed significant grossand microscopic lesion development of the ileum and colon innon-vaccinated, challenge control pigs (Group 4) compared to vaccinatedpigs regardless of dose (high, medium, and low) in Groups 1, 2, and 3(p<0.05). Average gross lesion scores of the vaccine-low dose colonswere not significantly different than challenge controls. Thevaccine-low dose group (Group 3) is the only treatment group thatreceived vaccine and had significant lesion development (gross andmicroscopic) in comparison to the strict control group which did notreceive a vaccination, placebo, or challenge throughout the study.

Clinical Scores

Clinical scores were recorded for each animal daily from day ofchallenge (day 21) to necropsy (day 42). Clinical scores were calculatedto obtain an average daily clinical score reflecting the severity andduration of sickness among treatment groups due to receiving a virulentchallenge of L. intracellularis. Average clinical scores for eachtreatment group are summarized in Table 8. TABLE 8 Treatment Group GroupIdentification Average Clinical Score 1 B3903 vaccine - high dose 3.1 a2 B3903 vaccine - medium dose 3.0 a 3 B3903 vaccine - low dose 3.0 a 4Challenge Control 3.0 a 5 Strict Control 3.0 a*Like letters denote no significant differences among average clinicalscores from the day of challenge to necropsy (p < 0.05).

Statistical analysis of clinical scores was accomplished by reviewingthe data in relation to treatment groups receiving average clinicalscores using 1-way ANOVA. There was no evidence of significantdifferences among non-vaccinated, challenge controls and strict controlscompared to the high, medium, or low dose vaccine groups.

Average Daily Weight Gains

Average daily weight gains (ADWG) were calculated from the time ofvaccine administration (day 0), to challenge administration (day 21), tothe termination of the study (day 42). Statistical analysis of weightgain differences (lbs.=pounds) between vaccine-high dose andnon-vaccine, challenge control treatment groups revealed evidence of asignificant difference from the time of challenge administration tonecropsy (p<0.05). The weight data is summarized in Table 9. TABLE 9Average ADWG ADWG ADWG Total Initial (day (day (lbs.) Treatment GroupWeight 0-21) 21-42) (Vaccination Group Identification (lbs.) (lbs.)(lbs.) to Necropsy) 1 B3903 vaccine 17.8 a 0.94 a 1.63 a 1.29 a highdose 2 B3903 vaccine 17.7 a 1.00 a 1.60 ab 1.30 a medium dose 3 B3903vaccine 18.0 a 0.94 a 1.48 ab 1.21 a low dose 4 Challenge 17.7 a 0.96 a1.45 b 1.21 a Control 5 Strict Control 17.7 a 0.94 a 1.63 ab 1.28 a*Like letters denote no significant differences between treatment groupsin ADWGs from day of vaccination to challenge and from day of challengeto necropsy (p < 0.05).

Seroversion (IFAT)

Serum samples were collected weekly from all test animals in eachtreatment group and tested for presence of anti-Lawsonia IgG antibodieson days 0, 7, 14, 21, 28, 35, and 42 of the study. Positive and negativeIFAT control samples were 100% accurate in all assays performed in thisstudy. On days 0 through 21 (3 weeks post vaccination), all test animalsin each treatment group were IFA negative. On day 28 (1 week postchallenge) of the study 1/15 (6.7%) test animals in the vaccine-highdose treatment group were IFA positive while all others tested IFAnegative. On day 35 (2 weeks post challenge) of the study, 4/15 (26.7%)pigs in the vaccine-high dose and medium dose treatment groups and 1/10(10%) pigs in the non-vaccine, challenge control treatment group wereIFA positive for Lawsonia antibodies. Both vaccine-low dose and strictcontrol treatment groups were IFA negative on day 35. On day 42 (3 weekspost challenge) of the study, 8/10 (80%) pigs in the challenge controlgroup, 6/15 (40%) pigs in the vaccine-medium dose group, 5/15 (33.3%)pigs in the vaccine-low dose group, and 3/15 (20%) pigs in thevaccine-high dose group were IFA positive. The strict control treatmentgroup was IFA negative at study termination (day 42).

Seroconversion data was analyzed using the Chi-square statistic. Forresults obtained in the strict control treatment group, a chi-squarestatistic was not computed due to the 100% negative responses found foreach test animal throughout the study. In treatment groups receiving avaccine or placebo, IFAT results were compared using Chi-squarestatistic with an estimation of exact p-value (Monte-Carlo). PositiveIFAT results obtained from challenge groups receiving a virulent pureculture challenge were significantly higher than those receivingvaccine-high dose and vaccine-low dose (p<0.05) at day 42 (3 weeks postchallenge) of the study.

Fecal Shedding of L. intracellularis (PCR)

Fecal swabs were collected weekly from all test animals in eachtreatment group and tested for the presence of L. intracellularis by PCRon days 0, 7, 14, 21, 28, 35, and 42 of the study. Positive and negativeDNA extraction and PCR reaction controls were 100% accurate for eachassay conducted in this study. All test animals in each treatment groupwere fecal PCR negative for L. intracellularis from day 0 (vaccination)to day 21 (challenge). Pigs in the strict control group remained fecalPCR negative for L. intracellularis in their feces throughout the study.Fecal shedding of L. intracellularis (fecal PCR positive) was evident invarious treatment groups each week after challenge inoculation. Theseresults are summarized in Table 10. TABLE 10 Day 42 Day 28 Day 35 (3weeks Treatment Group (1 week post (2 weeks post post GroupIdentification challenge) challenge) challenge) 1 B3903 vaccine 0/15(0%) a 2/15 (13.3%) a 0/15 (0%) a high dose 2 B3903 vaccine 6/15 (40%)5/15 (33.3%) a 0/15 (0%) a Medium dose ab 3 B3903 vaccine 4/15 (26.7%)8/15 (53.3%) 2/15 (13.3%) low dose ab ab ab 4 Challenge 4/10 (40%) b7/10 (70%) b 4/10 (40%) b Control 5 Strict Control 0/10 (0%) 0/10 (0%)0/10 (0%)*Like letters denote no significant difference among treatment groupsreceiving a challenge (p < 0.05).

Statistical analysis using Chi-square statistic with an estimation ofthe exact p-value (Monte Carlo) compared the positive responses in eachvaccine treatment group to the non-vaccine, challenge control grouponly. Results from the strict control group were withdrawn from groupcomparisons because each pig was 100% fecal PCR negative for L.intracellularis throughout the study. On day 28 (1 week post challenge),pigs receiving a high dose of vaccine had significantly less shedding ofL. intracellularis than non-vaccinated, challenged pigs (p=0.017). Onday 35 (2 weeks post challenge), pigs receiving a high dose (p=0.0024)and medium dose (p=0.041) of vaccine had significantly less shedding ofL. intracellularis in their feces than non-vaccinated challenged pigs.On day 42 (study termination), again, pigs receiving a high and mediumdose of vaccine (p=0.017) had significantly less shedding of L.intracellularis in their feces compared to the non-vaccinated,challenged pigs. Pigs in the low dose group were not significantly lessPCR positive than the challenge controls on any day post challenge.

L. intracellularis Tissue Colonization (PCR)

Polymerase Chain Reaction testing of tissue sections of the terminalileum, colon, tonsil, and mesenteric lymph node were performed afternecropsy (day 42 of the study). In the vaccine-high dose group, 1/15(6.7%) pigs were PCR positive for L. intracellularis colonization in thetonsil while all other treatment groups were PCR negative. Ileitis PCRtesting of mesenteric lymph tissue revealed 3/10 (30%) pigs in thenon-vaccinated, challenge controls were positive for L. intracellulariscolonization while all other treatment groups were PCR negative. IleitisPCR testing of mucosal scrapings of the terminal ileum revealed 4/10(40%) pigs in the challenge controls, 4/15 (26.7%) pigs in thevaccine-low dose group, 2/15 (13.3%) pigs in the vaccine-medium dosegroup, and 1/15 (6.7%) pigs in the vaccine-high dose group PCR positivefor L. intracellularis colonization. Ileitis PCR testing of the colonrevealed 3/10 (30%) pigs in the challenge controls, 5/15 (33.3%) pigs inthe vaccine-low dose group, 1/15 (6.7%) pigs in the vaccine-medium dosegroup, and 2/15 (13.3%) pigs in the vaccine-high dose group PCR positivefor L. intracellularis colonization. No evidence of L. intracellulariscolonization was seen in tissues of the strict control group.

Statistical analysis of Ileitis PCR positive results were compared amongtreatment groups using Chi-square statistic with Monte Carloapproximation of the exact p-value. Mesenteric lymph node PCR resultsindicated evidence of a significant difference among non-vaccinated,challenge controls and all treatment groups receiving a vaccinetreatment (p=0.054). No statistical significance was evident amongtreatment groups in L. intracellularis colonization in the ileum, colon,or tonsil at day 42 of the study.

Histology (IHC/H&E)

Sections 2 to 4 cm in length of tonsil, mesenteric lymph node, terminalileum, and colon were collected at necropsy (day 42) and placed in 10%buffered formalin for histological analysis. Lawsonia intracellulariswas not detected by IHC staining of tonsil sections in all treatmentgroups at necropsy. The strict control group was negative for L.intracellularis by IHC in all tissue samples taken at necropsy. Presenceof L. intracellularis and microscopic lesions associated with PPE werefound in the ileums of 9/10 (90%) pigs of the non-vaccinated, challengecontrols, 6/15 (40%) pigs of the vaccine-low dose group, 3/15 (20%) pigsof the vaccine-medium dose group, and 1/15 (6.7%) pigs of thevaccine-high dose group respectively. Microscopic lesions were evidentin the colon of 8/10 (80%) pigs in the challenge control group, 3/15(20%) pigs in the vaccine-low dose group, and 1/15 (6.7%) pigs in thevaccine-high dose group. The vaccine group receiving a medium dose wasIHC negative for L. intracellularis in the colon. Stained sections ofmesenteric lymph node produced only 1/10 (10%) pigs in the challengecontrol that had L. intracellularis. Positive IHC results in sections ofthe ileum and colon among treatment groups correlated well to thepresence of marked crypt hyperplasia demonstrated by H&E methodology.

Statistical analysis of quantitative IHC data was accomplished using1-way ANOVA and Kruskal-Wallis Rank Sum tests followed by specificcontrasts of p-values between the challenge group and strict controlgroups to each vaccine dosage group. Statistical analysis revealevidence of a significant lesion development due to L. intracellularisin the ileum and colon of the challenge control group compared tovaccinated pigs regardless of dose on day 42 of the study (p<0.001).Statistical significance was not evident in average IHC lesion scores ofthe ileum and colon in vaccinated animals receiving a high or mediumdose of vaccine compared to the strict control treatment group (p>0.05).Pigs receiving a low dose of vaccine had significantly higher meanmicroscopic lesion scores due to L. intracellularis in the ileumcompared to the strict control group (p<0.05).

Gross Scores

The ileum and colon of each test animal were scored at the time ofnecropsy (day 42) for gross lesions associated with PPE. Tissues fromtest pigs in the strict control were normal, did not contain lesions,and received average lesion scores of 1.1 (ileum) and 1.0 (colon).Tissues of test animals in the non-vaccinated, challenge control groupreceived the highest average gross lesion score for the ileum (3.6) andcolon (2.0) among treatment groups. Test pigs in the vaccine-low dosegroup received average gross lesion scores of 2.5 (ileum) and 1.5(colon). Vaccine-medium dose test pigs received average gross lesionscores of 1.5 (ileum) and 1.0 (colon). Tissues of the vaccine-high dosegroup received average gross lesion scores of 1.5 (ileum) and 1.2(colon).

Statistical analysis of average gross lesion scores among treatmentgroups was accomplished by using 1-way ANOVA. Average gross lesionscores indicated evidence of a significant difference between ileums innon-vaccinated, challenge control group and vaccine-medium and high dosegroups respectively (p<0.001). Evidence of a significant difference wasobserved among average gross ileum scores of the challenge controlcompared to the vaccine-low dose group (p<0.01). Average gross lesionscores of the colon were significantly higher in the challenge controlgroup compared to the vaccine-medium and high dose groups respectively(p<0.05). In addition, significant gross lesion development was observedin the ileum of the vaccine-low dose group compared to the strictcontrol group (p<0.001). No evidence of a statistical significance inaverage gross lesion scores (ileum and colon) were evident invaccine-medium and high dose groups compared to the strict controltreatment group.

Conclusions

This study demonstrated protection in 3-week-old pigs against PPE wasaccomplished when given an oral 2 mL dose of B3903 (Lyophilized) vaccinecontaining a minimum of 4.9 logs of live L. intracellularis per dose.Similar if not slightly better protection was evident in thevaccine-high dose treatment group that received 6.0 logs/dose 3 weeksprior to challenge. The low dose vaccine group (3.8 logs/dose) did notindicate adequate protection against a virulent pure cultureheterologous challenge compared to the test animals receiving a high ormedium dose of the experimental vaccine. Statistical differences wereevident among the vaccine-low dose group and non-vaccinated, challengecontrols regarding the severity of microscopic lesions in the ileum andcolon (p<0.001) and average gross lesion scores of the ileum (p<0.01).However, significant PPE lesions were observed in the ileum (microscopiclesions) and colon (gross lesions) of this group compared to the strictcontrol group that did not receive vaccine or challenge.

In summary, the data from this study demonstrated that: (1) the minimumprotective titer of a single oral administration of B3903 (Lyophilized)vaccine to 3 week old pigs is 4.9 logs/dose; (2) B3903 (Lyophilized)vaccine is efficacious against a virulent low passage pure culture L.intracellularis, heterologous isolate N101494; and (3) B3903(Lyophilized) vaccine aids in the reduction of gross and microscopiclesions, tissue colonization, and fecal shedding of L. intracellularisin vaccinated pigs compared to non-vaccinated pigs.

1. A virulent isolate of Lawsonia intracellularis, wherein said virulentisolate is Lawsonia intracellularis deposit isolate ATCC No. PTA-4927 ora Lawsonia intracellularis isolate having all of the identifyingcharacteristics of deposit isolate ATCC No. PTA-4927.
 2. An avirulentisolate of Lawsonia intracellularis, wherein said avirulent isolate isLawsonia intracellularis deposit isolate ATCC No. PTA-4926 or a Lawsoniaintracellularis isolate having all of the identifying characteristics ofdeposit isolate ATCC No. PTA-4926.
 3. A vaccine for the immunization ofan animal, comprising a pharmaceutically effective amount of a killedvirulent isolate of Lawsonia intracellularis, wherein said virulentisolate is the virulent isolate according to claim 1, and apharmaceutically acceptable carrier.
 4. A vaccine for the immunizationof an animal, comprising a pharmaceutically effective amount of theavirulent isolate of Lawsonia intracellularis according to claim 2, anda pharmaceutically acceptable carrier.
 5. The vaccine according to claim4 wherein the pharmaceutically effective amount of the avirulent isolateof Lawsonia intracellularis is about 10³ TCID₅₀ to about 10⁶ TCID₅₀ perdose.
 6. A vaccine for the immunization of an animal, comprising theavirulent isolate of Lawsonia intracellularis according to claim 2 andantigenic material from at least one of the following pathogens:Salmonella spp., Erysipelothrix spp., Haemophilus spp., Mycoplasma spp.,Leptospira spp., Clostridium spp., Streptococcus spp., Brachyspira spp.,circovirus, porcine reproductive and respiratory syndrome (PRRS) virus,swine influenza virus (SIV), transmissible gastro-enteritis (TGE) virus,parvovirus, or Escherichia coli; and a pharmaceutically acceptablecarrier.
 7. A vaccine for the immunization of an animal, comprising theavirulent isolate of Lawsonia intracellularis according to claim 2 andantigenic material from Salmonella choleraesuis, Erysipelothrix spp.transmissible gastro-enteritis (TGE) virus, Escherichia coli,Clostridium spp., and Brachyspira spp.; and a pharmaceuticallyacceptable carrier.
 8. A vaccine for the immunization of an animal,comprising the avirulent isolate of Lawsonia intracellularis accordingto claim 2 and antigenic material from Salmonella choleraesuis andErysipelothrix spp.; and a pharmaceutically acceptable carrier.
 9. Avaccine for the immunization of an animal, comprising the avirulentisolate of Lawsonia intracellularis according to claim 2 and antigenicmaterial from at least one of the following pathogens: Clostridium spp.,equine influenza virus (EIV), equine herpes virus (EHV), alphavirus, andWest Nile virus; and a pharmaceutically acceptable carrier.
 10. A methodfor stimulating the immune system of an animal to respond to animmunogenic antigen of pathogenic Lawsonia intracellularis, comprisingadministering to said animal an immunogenic composition containing theavirulent isolate of Lawsonia intracellularis according to claim
 2. 11.A method of immunizing an animal against porcine proliferativeenteropathy comprising administering a pharmaceutically acceptableamount of the avirulent isolate of Lawsonia intracellularis of claim 2,wherein the pharmaceutically effective amount of the avirulent isolateof Lawsonia intracellularis is about 10³ TCID₅₀ to about 10⁶ TCID₅₀ perdose.
 12. A method for making a vaccine for inducing an immune responseto Lawsonia intracellularis bacteria in an animal, comprising the stepsof: (a) incubating a Lawsonia intracellularis isolate according to claim1 in culture cells which are in suspension at an oxygen concentration ofless than about 10 percent to cultivate said bacteria; and (b) mixingsaid cultivated bacteria with a pharmaceutically acceptable carrier. 13.A method for making a vaccine for inducing an immune response toLawsonia intracellularis bacteria in an animal, comprising the steps of:(a) incubating a Lawsonia intracellularis isolate according to claim 2in culture cells which are in suspension at an oxygen concentration ofless than about 10 percent to cultivate said bacteria; and (b) mixingsaid cultivated bacteria with a pharmaceutically acceptable carrier. 14.The method according to claim 13 further comprising the step of killingsaid cultivated bacteria to prepare a vaccine containing killed Lawsoniaintracellularis.
 15. The method according to claim 13 further comprisingthe step of cultivating said bacteria for a sufficient time to producean attenuated isolate.
 16. A method for cultivating Lawsoniaintracellularis comprising: (a) infecting cultured cells with aninoculum comprising the virulent isolate of Lawsonia intracellularisaccording to claim 1, (b) incubating said infected cells at an oxygenconcentration of less than about 10 percent while maintaining saidinfected cells in suspension by agitation of said cells for a sufficientperiod of time to increase the production of said Lawsoniaintracellularis, and (c) harvesting at least a portion of said Lawsoniaintracellularis.
 17. The method according to claim 16 wherein the oxygenconcentration is in the range of about 0 percent to about 8 percent. 18.The method according to claim 17 wherein the oxygen concentration is inthe range of about 0 percent to about 3 percent.
 19. The methodaccording to claim 16 wherein said incubation occurs at a carbon dioxiderange of about 6 percent to about 10 percent.
 20. The method accordingto claim 16 wherein said cultured cells are selected from the groupconsisting of: HEp-2, McCoy, and IEC-18 cells.
 21. The method accordingto claim 20 wherein said IEC-18 cells are cultured on microcarriers. 22.The method according to claim 16 wherein the infected cells areincubated according to step (c) for a period of about 2 to about 10days.
 23. The method according to claim 16 further comprising a step (d)of cultivating said bacteria for a sufficient time to produce anattenuated isolate.
 24. The method according to claim 23 wherein thebacteria are cultivated according to step (d) for a period of about 150days to about 250 days.
 25. The method according to claim 24 furthercomprising a step (e) of lyophilizing said attenuated isolate.
 26. Themethod according to claim 25 wherein said lyophilizing comprises aprimary drying step and a secondary drying step.
 27. A lyophilizedattenuated isolate produced by the method according to claim
 26. 28. Amethod for the diagnosis of porcine proliferative enteropathy comprisingperforming an assay to detect antibodies to Lawsonia intracelluris in anon-human animal subject comprising: (a) contacting a sample from thesubject with the Lawsonia intracellularis isolate according to claim 1to form an antigen-antibody complex; and (b) detecting the formation ofsaid complex, wherein the presence of said complex correlates positivelywith a diagnosis of porcine proliferative enteropathy.
 29. A method forthe diagnosis of porcine proliferative enteropathy comprising performingan assay to detect antibodies to Lawsonia intracelluris in a non-humananimal subject comprising: (a) contacting a sample from the subject withthe Lawsonia intracellularis isolate according to claim 2 to form anantigen-antibody complex; and (b) detecting the formation of saidcomplex, wherein the presence of said complex correlates positively witha diagnosis of porcine proliferative enteropathy.
 30. The methodaccording to claim 28 wherein the assay comprises an assay selected fromthe group consisting of: radioimmunoassay, enzyme-linked immunosorbentassay, fluorescence immunoassay, and immunoelectrophoresis assay. 31.The method according to claim 29 wherein the assay comprises an assayselected from the group consisting of: radioimmunoassay, enzyme-linkedimmunosorbent assay, fluorescence immunoassay, and immunoelectrophoresisassay.
 32. A method for producing antisera against Lawsoniaintracelluris in a non-human animal comprising: (a) administering theLawsonia intracelluris isolate of claim 1 to a non-human animal in anamount effective to elicit an immune response; and (b) collectingantiserum or plasma containing antibodies to said Lawsoniaintracelluris.
 33. A method for producing antisera against Lawsoniaintracelluris in a non-human animal comprising: (a) administering theLawsonia intracelluris isolate of claim 2 to a non-human animal in anamount effective to elicit an immune response; and (b) collectingantiserum or plasma containing antibodies to said Lawsoniaintracelluris.